Antioxidants have become increasingly popular, namely in the biomedical field, because of their capacity to prevent the formation and the noxious activity of reactive oxygen species (ROS).
Plants and other photosynthetic organisms are particularly well adapted to resist the effect of ROS, especially to protect vital organelle photosynthetic membranes called thylakoids against oxidative damages and the noxious action of U.V. radiations.
Sunlight plays a much larger role in our sustenance than we may expect: all the food we eat and all the fossil fuel we use is a product of photosynthesis, which is the process that converts energy in sunlight to chemical forms of energy that can be used by biological systems. Photosynthesis is carried out by many different organisms, ranging from plants to bacteria. The best known form of photosynthesis is the one carried out by higher plants and algae, as well as by cyanobacteria and their relatives, which are responsible for a major part of photosynthesis in oceans. All these organisms convert CO2 (carbon dioxide) to organic material by reducing this gas to carbohydrates in a rather complex set of reactions. Electrons for this reduction reaction ultimately come from water, which is then converted to oxygen and protons. Energy for this process is provided by light, which is absorbed by pigments (primarily chlorophylls and carotenoids). Chlorophylls absorb blue and red light and carotenoids absorb blue-green light, but green and yellow light are not effectively absorbed by photosynthetic pigments in plants; therefore, light of these colors is either reflected by leaves or passes through the leaves.
Other photosynthetic organisms, such as cyanobacteria (formerly known as blue-green algae) and red algae, have additional pigments called phycobilins that are red or blue and that absorb the colors of visible light that are not effectively absorbed by chlorophyll and carotenoids. Yet other organisms, such as the purple and green bacteria (which, by the way, look fairly brown under many growth conditions), contain bacteriochlorophyll that absorbs in the infrared, in addition to in the blue part of the spectrum. These bacteria do not evolve oxygen, but perform photosynthesis under anaerobic (oxygen-less) conditions. These bacteria efficiently use infrared light for photosynthesis. Infrared is light with wavelengths above 700 nm that cannot be seen by the human eye; some bacterial species can use infrared light with wavelengths of up to 1000 nm. However, most pigments are not very effective in absorbing ultraviolet light (<400 nm), which also cannot be seen by the human eye. Light with wavelengths below 330 nm becomes increasingly damaging to cells, but virtually all light at these short wavelengths is filtered out by the atmosphere (most prominently the ozone layer) before reaching the earth. Even though most plants are capable of producing compounds that absorb ultraviolet light, an increased exposure to light around 300 nm has detrimental effects on plant productivity.
Photosynthetic pigments come in a huge variety: there are many different types of (bacterio)chlorophyll, carotenoids, and phycobilins, differing from each other in their precise chemical structure. Pigments generally are bound to proteins, which provide the pigment molecules with the appropriate orientation and positioning with respect to each other. Light energy is absorbed by individual pigments, but is not used immediately by these pigments for energy conversion. Instead, the light energy is transferred to chlorophylls that are in a special protein environment where the actual energy conversion event occurs: the light energy is used to transfer an electron to a neighboring pigment. Pigments and protein involved with this actual primary electron transfer event together are called the reaction center. A large number of pigment molecules (100–5000), collectively referred to as antenna, “harvest” light, capture photons, and transfer the light energy to the same reaction center. The purpose is to maintain a high rate of electron transfer in the reaction center, even at lower light intensities. The denomination P680 is assigned to the chlorophyll pigments of the reaction center PSII, because the pair of chlorophylls entering it composition absorbs light mostly at a 680 nm wavelength.
Many antenna pigments transfer their light energy to a single reaction center by having this energy transfer to another antenna pigment, and yet to another, etc., until the energy is “trapped” in the reaction center. Each step of this energy transfer must be very efficient to avoid a large loss in the overall transfer process, and the association of the various pigments with proteins ensures that transfer efficiencies are high by having appropriate pigments close to each other, and by having an appropriate molecular geometry of the pigments with respect to each other. An exception to the rule of protein-bound pigments are green bacteria with very large antenna systems: a large part of these antenna systems consists of a “bag” (named chlorosome) of up to several thousand bacteriochlorophyll molecules that interact with each other and that are not in direct contact with protein. Chlorophyll is used by all photosynthetic organisms as the link between excitation energy transfer and electron transfer. Of particular note is the rate with which these transfer reactions need to occur. As the lifetime of the excited state is only several nanoseconds (1 nanosecond (ns) is 10−9 s), after absorption of a quantum, energy transfer and charge separation in the reaction center must have occurred within this time period. Energy transfer rates between pigments are very rapid, and charge separation in reaction centers occurs in 3–30 picoseconds (1 picosecond (ps) is 10−12 s). Subsequent electron transfer steps are significantly slower (200 ps–20 ms) but, nonetheless, the electron transport chain is sufficiently fast that at least a significant part of the absorbed sunlight can be used for photosynthesis. The pigments have a specific organisation which should be preserved upon isolation and purification of thylakoids if the maintenance of the function of the latter is sought.
In many systems the size of the photosynthetic antenna is flexible, and photosynthetic organisms growing at low light (in the shade, for example) generally will have a larger number of antenna pigments per reaction center than those growing at higher light intensity. However, at high light intensities (for example, in full sunlight) the amount of light that is absorbed by plants exceeds the capacity of electron transfer initiated by reaction centers. Plants have developed means to convert some of the absorbed light energy to heat rather than to use the absorbed light necessarily for photosynthesis. However, in particular the first part of photosynthetic electron transfer in plants is rather sensitive to overly high rates of electron transfer, and part of the photosynthetic electron transport chain may be shut down when the light intensity is too high; this phenomenon is known as photoinhibition.
The initial electron transfer (charge separation) reaction in the photosynthetic reaction center sets into motion a long series of redox (reduction-oxidation) reactions, passing the electron along a chain of cofactors and filling up the “electron hole” on the chlorophyll, much like in a bucket brigade. All photosynthetic organisms that produce oxygen have two types of reaction centers, named photosystem II and photosystem I (PS II and PS I, for short), both of which are pigment/protein complexes that are located in specialized membranes called thylakoids. In eukaryotes (plants and algae), these thylakoids are located in chloroplasts (organelles in plant cells) and often are found in membrane stacks (grana). Prokaryotes (bacteria) do not have chloroplasts or other organelles, and photosynthetic pigment-protein complexes either are in the membrane around the cytoplasm or in invaginations thereof (as is found, for example, in purple bacteria), or are in thylakoid membranes that form much more complex structures within the cell (as is the case for most cyanobacteria).
All the chlorophyll in oxygenic organisms is located in thylakoids, and is associated with PS II, PS I, or with antenna proteins feeding energy into these photosystems. PS II is the complex where water splitting and oxygen evolution occurs. Upon oxidation of the reaction center chlorophyll in PS II, an electron is pulled from a nearby amino acid (tyrosine) which is part of the surrounding protein, which in turn gets an electron from the water-splitting complex. From the PS II reaction center, electrons flow to free electron carrying molecules (plastoquinone) in the thylakoid membrane, and from there to another membrane-protein complex, the cytochrome b6f complex. The other photosystem, PS I, also catalyzes light-induced charge separation in a fashion basically similar to PS II: light is harvested by an antenna, and light energy is transferred to a reaction center chlorophyll, where light-induced charge separation is initiated. However, in PS I electrons are transferred eventually to NADP (nicotinamide adenosine dinucleotide phosphate), the reduced form of which can be used for carbon fixation. The oxidized reaction center chlorophyll eventually receives another electron from the cytochrome b6f complex. Therefore, electron transfer through PS II and PS I results in water oxidation (producing oxygen) and NADP reduction, with the energy for this process provided by light (2 quanta for each electron transported through the whole chain).
Electron flow from water to NADP requires light and is coupled to generation of a proton gradient across the thylakoid membrane. This proton gradient is used for synthesis of ATP (adenosine triphosphate), a high-energy molecule. ATP and reduced NADP that resulted from the light reactions are used for CO2 fixation in a process that is independent of light. CO2 fixation involves a number of reactions that is referred to as the Calvin-Benson cycle. The initial CO2 fixation reaction involves the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which can react with either oxygen (leading to a process named photorespiration and not resulting in carbon fixation) or with CO2. The probability with which RuBisCO reacts with oxygen vs. with CO2 depends on the relative concentrations of the two compounds at the site of the reaction. In all organisms CO2 is by far the preferred substrate, but as the CO2 concentration is very much lower than the oxygen concentration, photorespiration does occur at significant levels. To boost the local CO2 concentration and to minimize the oxygen tension, some plants (referred to as C4 plants) have set aside some cells within a leaf (named bundle-sheath cells) to be involved primarily in CO2 fixation, and others (named mesophyll cells) to specialize in the light reactions: ATP, CO2 and reduced NADP in mesophyll cells is used for synthesis of 4-carbon organic acids (such as malate), which are transported to bundle sheath cells. Here the organic acids are converted releasing CO2 and reduced NADP, which are used for carbon fixation. The resulting 3-carbon acid is returned to the mesophyll cells. The bundle sheath cells generally do not have PS II activity, in order to minimize the local oxygen concentration. However, they retain PS I, presumably to aid in ATP synthesis.
Thylakoid organization is very sophisticated in order to extract the energy from light, and to transfer this energy to a proper location, and/or dissipate the same. The transfer is rendered possible and efficient by separating electrical charges and a high capacity to regenerate a neutral electrical state, ready for undertaking again a change in charges (Blankenship et al. 1998).
The electron transfer between the above five main components is extremely rapid: the transfer from an activated P680 to pheophytin takes less than one picosecond. The electron transfer stops when all the pigments return to a neutral electrical charge, ready to undertake a new cycle.
Electrons are finally directed to a coupling factor to reduce NADPH, necessary in ATP synthesis, which will serve in sugar synthesis.
The term “thylakoids” is used hereinbelow and means to cover organized photosynthetic membrane components obtained from photosynthetic organisms, eucaryotic and prokarytotic. When the organism has chloroplasts, the thylakoids comprise the following membrane constituents: PSII, cytochromes b6 and f, PSI and the coupling factor. Where thylakoids integrity and functionality has been tested from plant material, it has been measured between two reference points: proximal to PSII and distal to the coupling factor. For certain applications, thylakoids do not need to be active although they are apparently integral. Such thylakoids are performing and at least as stable as any other antioxidant. Therefore, “active thylakoids” means thylakoids having the capacity to activate upon hydration, as opposed to inactive thylakoids which are integral but which have been actively or passively inactivated. In this case, the reaction center is inactive although thylakoids structure is substantially preserved. The “inactive” thylakoids are therefore suitable antioxidants although they do not have the same dynamism nor do they have the same capability to regenerate, or the same capacity to respond to ROS as the active/activable form.
Photosynthesis comprises two fundamental processes that can be summarized in the two following reactions:
CO2+ATP+NADPH+H+→(CH2O)n+ADP+Pi+NADP+  (2)
During the first reaction in the presence of light, protons are taken from chloroplast water to produce ATP. The second reaction consists in using NADPH and ATP in a series of reactions that lead to the reduction of carbon anhydride in glucides, mainly starch. These two reactions occur simultaneously; products formed by process (1) are directed into the reaction of process (2). Globally, the photosynthesis results into the production of sugars in the form of starch and sucrose and energy under the form of ATP molecules:

Light activation follows a certain pathway in the thylakoids. Light is first collected by light antenna (LHCII), and the energy is directed to reaction center (PSII) and, finally to PSI which also has an independent light collector (LHCI). Thylakoids have for functions to collect light and to transfer light energy to a proper location for further photosynthesis. The synthesis of ATP and of sugars does not take place in the thylakoids but in other chloroplast compartments.
Chlorophylls are the main active pigments. The carotenoids have more than one role, depending on their location. A first role is as light collectors, which results in energy transfer from carotenoids to chlorophylls. A second role is as photoprotectors, this time the energy transfer occurring in an opposite direction between chlorophylls and carotenoids. Carotenoid singlet state has more energy that a singlet chlorophyll while, on the opposite, carotenoid triplet state has less energy than triplet chlorophylls. The energy states having a natural tendency to go from a high to a low energy level, one will appreciate that the singlet carotenoid mostly acts as a light collector passing light energy to a singlet chlorophyll molecule while the triplet chlorophyll will readily transfer its energy to the triplet carotenoid, when the latter acts as a photoprotector in the reaction center. Carotenoids take different configurations upon associating with antenna or reaction center, which configuration may be responsible for their energy state upon activation. A “cis” configuration is associated with photoprotection in the reaction center. An “all-trans” configuration is associated with the light collector function of the antenna.
The transfer of energy is efficient only in conditions in which the pigments are very close to each other and in a specific organisation. It is therefore very important not to disturb the natural organisation of the pigments, keeping the membranes in an integral state, if one wants to purify active or fully activable thylakoids.
One advantage of recovering intact thylakoids is found in their capacity to handle ROS. Such ROS are intended to cover free radicals (including super oxides), as well as non-radical oxidants such as singlet oxygen (1O2) and peroxides. A good review of the definition and origin of these species is found in the international publication WO 94/13300. The contents of all the references cited hereinabove and below are incorporated herein by reference.
Free radicals are atoms, ions, or molecules that contain an unpaired electron. Free radicals are usually unstable and exhibit short half-lives. Elemental oxygen is highly electronegative and readily accepts single electron transfers from cytochromes and other reduced cellular components; a portion of the O2 consumed by cells engaged in aerobic respiration is univalently reduced to superoxide radical (.O2−). Sequential univalent reduction of .O2− produces hydrogen peroxide (H2O2), hydroxyl radical (.OH), and water.
Free radicals can originate from many sources, including aerobic respiration, cytochrome P-450-catalyzed monooxygenation reactions of drugs and xenobiotics (e.g., trichloromethyl radicals, CCl3, formed from oxidation of carbon tetrachloride), and ionizing radiation. For example, when tissues are exposed to gamma radiation, most of the energy deposited in the cells is absorbed by water and results in scission of the oxygen-hydrogen covalent bonds in water, leaving a single electron on hydrogen and one on oxygen creating two radicals H and OH. The hydroxyl radical, .OH, is the most reactive radical known in chemistry. It reacts with biomolecules and sets off chain reactions and can interact with the purine or pyrimidine bases of nucleic acids. Indeed, radiation-induced carcinogenesis may be initiated by free radical damage. Also for example, the “oxidative burst” of activated neutrophils produces abundant superoxide radical, which is believed to be an essential factor in producing the cytotoxic effect of activated neutrophils. Reperfusion of ischemic tissues also produces large concentrations of oxyradicals, typically superoxide. Moreover, superoxide may be produced physiologically by endothelial cells for reaction with nitric oxide, a physiological regulator, forming peroxynitrite, ONOO− which may decay and give rise to hydroxyl radical, .OH. Additional sources of oxyradicals are “leakage” of electrons from disrupted mitochondrial or endoplasmic reticular electron transport chains, prostaglandin synthesis, oxidation of catecholamines, and platelet activation. Many free radical reactions are highly damaging to cellular components; they crosslink proteins, mutagenize DNA, and peroxidize lipids. Once formed, free radicals can interact to produce other free radicals and non-radical oxidants such as singlet oxygen (1O2) and peroxides.
Singlet oxygen is a particularly noxious compound involved in the initiation or in the perpetuation of many diseases or disorders. The singlet oxygen is also involved in the degradation of protein like chlorophylls. This is why a photoprotection conferred by the presence of carotenoids becomes important. Carotenoids protect the chlorophyll life and activity, they further protect the integrity of the membranes by preventing protein denaturation. Carotenoids are capable of capting the energy of triplet chlorophyll molecules; they become triplet carotenoid molecules, which regenerate themselves while dissipating heat thereby avoiding the accumulation of a triplet chlorophyll, and minimizing the chances to degrade the chlorophyll.
However, in the presence of excess light, damage may occur, which may originate from the formation of chlorophyll in “triplet state”. In a triplet state two electrons in the outer shell have identical rather than opposite spin orientation. This triplet chlorophyll readily reacts with oxygen, leading to the very reactive singlet oxygen, which can damage proteins. To counter this damaging reaction, carotenoids are usually present in close vicinity to chlorophylls. Many carotenoids efficiently “quench” triplet states of chlorophyll, thus avoiding formation of singlet oxygen. Chlorophyll in its free form is very toxic in the light in the presence of oxygen, because a close interaction with carotenoids is not always available under such circumstances. Therefore, all chlorophyll in a cell in aerobic organisms is bound to proteins, generally with carotenoids bound to the same protein.
A major difficulty in measuring enzyme kinetics at relatively short time scales (less than 1 ms) is that “traditional” enzyme reactions require a mixing of substrate and enzyme, which usually takes a relatively long time. Kinetic analysis of light-driven reactions such as photosynthetic electron transport have a great advantage in this respect: reactions can be triggered simply by a light pulse, which can be even shorter than 1 ps. Moreover, many of the components participating in electron transfer have different absorption spectra depending on whether they are in the oxidized or reduced form. Using laser spectroscopy methods or more standard optical spectroscopy, it is relatively simple to follow the electron around on a timescale between 1 ps and several ms. The primary charge separation occurs in several ps, and reactions become gradually slower as they involve components that are further away from the reaction center. Because of the fast speed of early reactions, the electron and the “electron hole” are physically separated rapidly by a large distance (the electron generally has traveled about 2 nm to the other side of the membrane within 1 ns after charge separation), so that back reactions (charge recombinations) are not favorable anymore. Unpaired electrons on reactants that are transiently formed during redox reactions involving transfer of a single electron in many instances can be detected using electron paramagnetic resonance (EPR) and derived techniques (including ENDOR, electron nuclear double resonance, and ESEEM, electron spin echo envelope modulation). Many of these techniques can be used to kinetically follow redox reactions, and provide detailed information regarding electron spin distributions etc. Therefore, photosynthetic membranes and reaction centers have a prominent place as experimental systems in biochemistry and biophysics.
The anti-oxidative potential of a compound such as chlorophyll is exemplified in equation (1)3Chl*+3O2→Chl+1O2*  (1)
Chlorophyll that has been excited into presence of oxygen becomes in a triplet state (3Chl*), and disactivates to return to a fundamental state by producing singlet oxygen (a noxious species) in cells. Plants have found an efficient means by which they can solve the problem of overproduction of singlet oxygen. The plants transfer the chlorophyll energy to another pigment which has an inferior energy state. That pigment called carotenoid (equation 2) is abundant in plants.3Chl*+Car→Chl+3Car*  (2)
Although triplet chlorophyll has more energy than a corresponding carotenoid, the converse is true for the singlet state. As shown in equations 3a and 3b, an activated singlet carotenoid transfers its energy to a chlorophyll molecule which becomes activated in a singlet state.Car+energy→1Car*  (3a)1Car*+Chl→Car+1Chl*  (3b)
Carotenoids in a triplet state desactivates without forming a noxious oxygen species. Equation 4 shows that carotenoids inactivate by returning to a fundamental state and by heat dissipation.3Car*→Car+Heat  (4)
It appears that it is important not to produce ROS to preserve the properties of the pigments in an extract, but it is also important to remove those ROS that may be generated during isolation. For achieving this, we have given favor to a way to reverse the equilibrium of equation 1. Consequently, the converse equation 1 is found in equation 5.Chl+1O2*→3Chl*+3O2  (5)
To avoid reversal of equation 5, activated triplet chlorophyll molecule needs to be in close contact with a carotenoid in its fundamental state, which takes the transferred energy and dissipates the latter as heat. This way the reversibility of equation 5 is restricted insofar as chlorophyll and carotenoid pigments can be found in very close proximity so as to transfer to one another their energy.
From the above equation 5, it is apparent that, to obtain an extract that is optimally active, it is preferable to take every possible measures to maintain both pigments (chlorophyll and carotenoid) in their fundamental state. Isolated carotenoids, e.g. carotenoids not organized in thylakoid structures, would not be capable of an efficient quenching of triplet chlorophyll molecules. The advantage of having organized pigments is that the extract will retain the dynamism of natural thylakoid membranes, which confers to them the capacity to capture ROS, to transfer the energy and to return to a state capable of undertaking new activation cycles again. This dynamism and capacity to regenerate is unique to organized pigments. It is important to mention that the above reactions are spontaneously produced and this, in absence of light. This observation is important from a therapeutic point of view, because internal administration of a thylakoid extract would preclude the presence of light.
Thylakoids having optimized configuration and carotenoid proportions will retain full activity especially toward ROS. Such an antioxidant will be useful to reduce the expression of diseases or disorders that involve the production of ROS. Such diseases or disorders can be those with an etiology related to inflammation, cancer and contact with radiations. Such diseases or disorders comprise those affecting Skin: such as bums, solar radiation, psoriasis, dermatitis; Brain: such as trauma, stroke, Parkinson, neurotoxins, dementia, Alzheimer; Joints: such as rheumatoid arthritis and arthrosis; Gastrointestinal tract: such as diabetes, pancreatitis, endotoxin liver injury, ischemic bowel; Eye: such as cataractogenesis, retinopathy, degenerative retinal damage; Vessels: such as atherosclerosis and vasculitis; Erythrocytes: such as Fanconi anemia, malaria; Heart: such as coronary thrombosis; Lung: such as asthma, COPD; Kidney: such as transplantation, glomerulonephritis; Multiorgan: such as inflammation, cancer, ischemia-reflow states, drug toxicity, iron overload, nutritional deficiencies, alcohol toxicity, radiation, ageing, amyloid diseases and toxic shock. The literature related to the involvement of ROS in some diseases is the following:
Skin:BurnYoun, 1992Solar RadiationGolan, 1994PsoriasisLange, 1998 a, bDermatitisPolla, 1992Brain:TraumaJuurlink, 1998StrokeEl Kossi, 2000ParkinsonEbadi, 1996NeurotoxinsFoler, 2000AlzheimerSmith 2000Joints:Rheumatoid arthritisCimen, 2000Gastrointestinal tract:DiabetesGerber, 2000PancreatitisSakorafas, 2000Endotoxin liver injuryMcGuire, 1996Ischemic bowelLai, 2000Eye:CataractogenesisEaton, 1994Retinopathy of prematurityHardy, 2000Degenerative retinal damageCastagne, 2000Vessels:AtherosclerosisSingh, 1997Erythrocytes:AnemiaAnastassopoulou, 2000MalariaGinsburg, 1999Heart:Coronary thrombosisChen, 1995Lung:AsthmaMontuschi, 1999COPDMontuschi, 2000Kidney:Glomerulonephritis:Barros, 2001Multiorgan:TransplantationJonas, 2000InflammationEl-Kadi, 2000CancerPrior, 2000IschemiaLewen, 2000Drug toxicitySinha, 1990Iron overloadKarbownik, 2000Nutritional deficienciesOlszewski, 1993Alcohol toxicityLieber, 1997AgeingCadenas, 2000RadiationBednarska, 2000Amyloid diseasesFloyd, 1999.
Besides therapeutic applications, it has been found that the thylakoids of this invention may advantageously replace chloroplasts-derived compositions of the art that have been tested as biosensors or biofilters or bioreactors. The art in the field teaches these specific uses, but the chloroplasts-derived compositions lack stability and degrade very rapidly, which renders these uses unpractical from a commercial point of view. Therefore, a stable and dynamic thylakoid extract could advantageously substitute for these non-performing chloroplasts-derived compositions.
Biosensors:
Detection of toxic products is valuable for evaluating environmental risks associated with the presence of contaminants. Valid bioassays would normally involve living organisms and would fulfil the following minimal conditions:    i) they should be representative of the natural environment,    ii) they should reproducible,    iii) they should be reliable so as to provide no or almost no false results; and    iv) they should be sensitive.
Toxicity detection should also provide enough flexibility for analyzing different types of contaminated samples. Toxicity should be ideally monitored and sensed in real time fashion. Toxicity detection finds application in at least three industrial sectors: paper industry, contaminated soil analysis and agriculture. In all these instances, information is needed on the presence of contaminant in order to rapidly correct an undesirable situation.
A major problem encountered with the actual technologies to sense toxic products is in the long delay of obtention of the results of biotests from 48 to 96 hours, when using organisms like trout or Daphnea magna. A good detecting device would be one distinguishing from the available conventional biotests by the use of material which would allow measurements of a contaminating potential of an effluent in real time and continuously. Although some biodetectors are commercially sold, which measure fluorescence generated by plant photosynthetic activity, a system that would permit measurement of electrical charges induced by the presence of light, and modulated by the presence of contaminants would be ideal. This technology would be much cheaper than the fluorimetric technology. It is believed that a technique which would evaluate the photosynthetic activity on a total thylakoid material would be preferable over fluorometric methods which measure the activity of a specific proteic complex, namely the PSII. A device comprising thylakoid material would therefore have the advantage of measuring the toxicity in a larger spectrum of action. Such as detecting device would measure the number of electrons produced with a given light intensity. A current (Epmax) obtained after a few seconds should be proportional to the photosynthetic activity of the thylakoids. If the photocurrent is plotted against the concentration of contaminants, a typical sigmoidal should be obtained, upon which an estimated EC50 should be deduced.
A photocurrent has been already measured by Allen and Crane in 1976. It has been found that electron transport constituted a reliable and representative measure of global photosynthetic activity and of the physiological health of a plant. From the work of Allen and Crane, it is conceivable that an extract that would have a great stability, along with a dynamism and capacity to regenerate its responsive state to contaminants and light would be highly preferable over the known devices. A detector would measure the number of electrons produced at a given light intensity. A maximal photo-current value (Ipmax) obtained after a few seconds is proportional to the photosynthetic activity of the thylakoid membranes. If one plots Ipmax v. the concentration of a photosynthetic inhibitor (a contaminant or a pollutant) present in the photoconversion chamber, a typical sigmoidal curve is obtained. The inhibitor potency can be easily evaluated (IC50).
A detecting device would comprise: a white light source, a photoconversion chamber receiving two electrodes, a detecting means for measuring electrical currents induced by light and computer means for collecting and processing data (electric currents). A liquid sample comprising a toxic agent, a contaminant or a pollutant to be identified or measured, is contacted with a thylakoid membrane extract. Once the mixture introduced in the chamber, a brief illumination is applied (less than one minute). The device or apparatus may be conceived to process and analyze a plurality of samples simultaneously.
Biofilters/Bioreactors:
Because the photosynthetic apparatus in plants is capable of not only capturing photons, but also of capturing and accumulating molecules having affinity for its components, it is contemplated that the present extract would also have the same capacity as the plant itself. Moreover, since some of the captured molecules may be processed, the present extract would act as a bioreactor. The molecules susceptible to be captured are, for example, herbicides, insecticides, fungicides, urea, ions and heavy metals as well as gas like O3, CO, H2S, NO, CO2, O2 . . . The biofilter of this invention would be versatible and would be resistant to temperature variations.
There is no existing practical process in the art teaching how to recover intact functional thylakoids, capable of retaining activity for a practical amount of time.
It is obvious from the above that the plants have a great natural capacity to manage with threatening situations. The thylakoids are particularly adapted to resist and adapt to such extreme situations.
The U.S. Pat. No. 4,698,360 describes a plant extract comprising pro-anthocyanidins useful as free-radical scavengers. The process of making this extract comprises the following steps:    a) the obtention of a coarse powder of maritime pine bark;    b) its extraction in boiling water;    c) a separation of liquids from solids;    d) cooling the liquids to ambient temperature;    e) a filtration;    f) a “salting-out” precipitation to remove undesirable matter;    g) extracting active ingredients into ethyl acetate;    h) drying the organic phase;    i) resuspending the solids and reprecipitating the active ingredients with chloroform; and    j) resuspending the solids before advanced purification.
This reference is concerned with the isolation of a specific type of active ingredient, and not with the preparation of thylakoids that would contain a major portion of its photosynthetic components, in other words wherein pigments would not be separated from each another.
This reference is indeed typical of the overall teachings in the general art which the present invention pertains to. The prior art relates systematically to the isolation of one or more given plant components, and not to the isolation of intact thylakoids comprising a major portion of their constituents preserved in an integral and functional state.
Glick et al. (1985) in Planta 164: 487–494 describe the variations in stoichiometric ratios of photosystems II and I (PSII/PSI) when peas are submitted to different types of light. The electron transport capacity of PSI and PSII in the presence of indicators such as 2,5-dimethyl-p-benzoquinone and NADP, which are indicators specific for PSII and PSI, respectively. Although green light is used, which is a non-activating light environment, it is not used to condition the plant in a process which aims at isolating intact and activable thylakoids. The reference essentially relates to the study of the composition of chloroplasts and not the preservation of thylakoids activity in function of a given light quality and intensity. The plants are rather conditioned in different lights that are depleted or enriched in red wavelengths. This reference is not concerned with the fact that the photosynthetic pigments should be kept close to each other so as to favorise the energy transfer between chlorophylls and carotenoids and to favorise free-radicals capture. Thus the conditions leading to the isolation of photosynthetic pigments in their natural state in thylakoids are not specifically taught and met with in this reference.
Mason et al. (1991) in Plant Physiol. 97: 1576–1580 teach a method for isolating chloroplasts, which makes use of a step of forced passage of a plant suspension through a 27-needle at a flow speed of 0.5 ml per second, rather than using a dispersion step by homogenization. The plant solution comprises a buffer having a pH 7.5 and comprising 0.3 M sorbitol. The preparation that has been forced through the needle is centrifuged in a Percoll gradient and the chloroplasts are separated from other constituents, including thylakoids. This process is therefore different from the present process which aims essentially at the recovery of thylakoids using quite simple steps and reactants, which present process being also easy to scale up. The light conditions are not mentioned in this reference. Further, the conditions to keep chloroplasts integral are obviously not the same as conditions to disintegrate chloroplasts. In the present process, the chloroplasts are disintegrated but thylakoids membranes are recovered substantially intact. This reference therefore cannot teach the present invention.
The Canadian patent application 2,110,038 describes a process of stabilizing plant extracts. These extracts are however cell fluids or juices and not thylakoid membranes. There is no mention in this reference of withdrawal of water as a natural electron donor from the membranes, for the purpose of stabilizing thylakoids.
In view of the foregoing, no practical process has been taught in the art, that would lead to the isolation of intact and functional thylakoid membranes. There is further no teaching of conditions for stabilizing thylakoid components. There is finally no teaching of the use of isolated thylakoid membranes to scavenge cell components from ROS.
There is therefore an open challenge in developing a process for obtaining active thylakoids that remain integral and, optionally activable, for an acceptable amount of time and which, upon reactivation are capable of acting as an antioxidant by their ROS scavenging activity. Although an increasing body of literature is available on photosystem components, nobody has published a practical process wherein the conditions of isolation and preservation of thylakoid activity are taught.
Moreover, since free radicals may be responsible for the degradation of many cell components, it is expected that their capture would protect other plant constituents. The present process would therefore produce an improved yield of plant components other than thylakoids.
Because there is a demand for powerful antioxidants, particularly in the pharmaceutical field, a process providing any such antioxidants, as well as the antioxidants per se capable of a good potency as well as of a sustained activity, would be greatly appreciated. Further, there is a demand for biological material useful as sensors or detectors, captors or filters, bioreactors or biological molecule producers.